Transposition, amplification, and divergence in the origin of the DNF15 loci, a polymorphic repetitive sequence family on chromosomes 1 and 3

The loci DNF15S1 and DNF15S2 are members of a small repetitive sequence family at discrete chromosomal locations, namely, 1p36 and 3p21, respectively. Studies of the structure, arrangement, and interrelations of the family suggest that the single copy on chromosome 3 is the original member and that this gave rise to the several members on chromosome 1 by transposition, partial duplication, and amplification. Several restriction fragment length polymorphisms have been discovered at the DNF15S1 locus and these have been assigned to the different subfamilies of the repeat at this locus. The existence of these RFLPs, and the nonallelic restriction site variation also found in this sequence family, suggests that transposition and amplification occurred as discrete events. We sequenced across the ancient junction between chromosomes 1 and 3 and noted features which might explain the mechanics of the transposition and amplification events.     

Incidence of AIDS and excess of mortality associated with HIV in haemophiliacs in the United Kingdom: report on behalf of the directors of haemophilia centres in the United Kingdom.

OBJECTIVE--To estimate the cumulative incidence of AIDS by time since seroconversion in haemophiliacs positive for HIV and to examine the evidence for excess mortality associated with HIV in those who had not yet been diagnosed as having AIDS. DESIGN--Analysis of data from ongoing national surveys. SETTING--Haemophilia centres in the United Kingdom. PATIENTS--A total of 1201 men with haemophilia who had lived in the United Kingdom during 1980-7 and were positive for HIV. INTERVENTION--None. END POINTS--Diagnosis of AIDS; death in those not diagnosed as having AIDS. MEASUREMENTS AND MAIN RESULTS--Estimation of cumulative incidence of AIDS and number of excess deaths in seropositive patients not diagnosed with AIDS. Median follow up after seroconversion was 5 years 2 months. Eight five patients developed AIDS. Cumulative incidence of AIDS five years after seroconversion was 4% among patients aged less than 25 at first test positive for HIV, 6% among those aged 25-44, and 19% among those aged greater than or equal to 45. There was little evidence that type or severity of haemophilia or type of factor VIII or IX that had caused HIV infection affected the rate of progression to AIDS. Mortality was increased among those who had not been diagnosed as having AIDS, especially among those with "AIDS related complex." Thirteen deaths were observed among 36 patients diagnosed as having AIDS related complex against 0.65 expected, and 34 deaths in 1080 other patients against 22.77 expected; both calculations were based on mortality rates observed in haemophiliacs in the United Kingdom in the late 1970s. CONCLUSIONS--Rate of progression to AIDS depended strongly on age. There is a substantial burden of fatal disease among patients positive for HIV who have not been formally diagnosed as having AIDS.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Apparent regression of the CGG repeat in FMR1 to an allele of normal size

The fragile X syndrome is the result of amplification of a CGG trinucleotide repeat in the FMR1 gene and anticipation in this disease is caused by an intergenerational expansion of this repeat. Although regression of a CGG repeat in the premutation range is not uncommon, regression from a full premutation (>200 repeats) or premutation range (50–200 repeats) to a repeat of normal size (<50 repeats) has not yet been documented. We present here a family in which the number of repeats apparently regressed from approximately 110 in the mother to 44 in her daughter. Although the CGG repeat of the daughter is in the normal range, she is a carrier of the fragile X mutation based upon the segregation pattern of Xq27 markers flanking FMR1. It is unclear, however, whether this allele of 44 repeats will be stably transmitted, as the daughter has as yet no progeny. Nevertheless, the size range between normal alleles and premutation alleles overlap, a factor that complicates genetic counseling.     

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.     

Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K_is ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C₁---C₂ double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k_inacts ranging from 4.4 × 10⁻⁴ to 1.90 x 10⁻³ s⁻¹. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T_1/2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC₅₀ values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.     

The 5-55 single-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor.

An analogue of the BPT1 folding intermediate that contains only the disulphide bond between Cys-5 and Cys-55 has been prepared by mutation of the other four Cys residues to Ser. On the basis of its circular dichroism and 1H-nuclear magnetic resonance spectra and its electrophoretic mobility, this intermediate is shown to be at least partially folded at low temperatures. This probably accounts for several of the unique properties of this intermediate observed during folding.